The chemistry-biology interface is experiencing a renaissance with the advent of many fluorescence based techniques for studying cell and tissue level events (Lichtman et al., 2005, Nat. Methods 2: 910-919; Hein et al., 2008, Proc. Natl. Acad. Sci. USA 105: 14271-14276; Patterson et al., 2010, Annu. Rev. Phys. Chem. 2010 61: 345-367; Huang et al., 2009, Annu. Rev. Biochem. 78: 993-1016). Therefore, there is an increasing need for new fluorescent molecules with uniquely tailored properties for biological imaging (Grimm et al., 2013, Prog. Mol. Biol. Transl. 113: 1-34; Ramil and Lin, 2013, Chem. Commun. 49: 11007-11022; Shih et al., 2014 Curr. Opin. Chem. Biol. 21: 103-111). Among them are photoactivatible probes which allow for the possibility of precise spatial and temporal control with minimal physical and chemical intervention. There are many hurdles and requirements for the development of photoactivatable fluorophores and very few irreversible intracellular photoactivation mechanisms have been reported beyond traditional photoswitching and photodecaging strategies (Kobayashi et al., 2012, J. Am. Chem. Soc. 134: 11153-11160; Lord et al., 2008, J. Am. Chem. Soc. 130: 9204-9205; Lim and Lin, 2011, Accounts Chem. Res. 44: 828-839; Ramil and Lin, 2014, Curr. Opin. Chem. Biol. 21: 89-95; Faal et al., 2015, Mol. bioSystems 11: 783-790; Chozinski et al., 2014, FEBS letters 588: 3603-3612; Ueno et al., 2004, J. Am. Chem. Soc. 126: 14079-14085; Yu et al., 2011, J. Am. Chem. Soc. 133: 11912-11915; An et al., 2013, Org. Lett. 15: 5496-5499; Vaughan et al., 2012, Nat. Methods 9: 1181-1184). New photoactivation concepts and mechanisms would expand the fluorescent probe toolbox providing new tools for investigating biological systems.
Photoactivatable fluorescent probes are powerful tools for studying biological system due to the high spatial and temporal control afforded by light. Photoactivatable probes can be genetically encoded proteins (Ludyanov et al., 2005, Rev. Mol. Cell Biol. 6: 885-891; Welman et al., 2010, J. Biol. Chem. 285: 11607-11616; Verkhusha and Sorkin, 2005, Chem. Biol. 12: 279-285; Patterson and Lippincott-Schwartz, 2004, Methods 32: 445-450; Shcherbakova and Verkhusha, 2014, Curr. Opin. Chem. Biol. 20: 60-68; Adam and Berardozzi, 2014, Curr. Opin. Chem. Biol. 20: 92-102) or small molecules (Lacivita et al., 2012, Curr. Med. Chem. 19: 4731-4741; Mernandez-Suarez and Ting, 2008, Nat. Rev. Mol. Cell Biol. 9: 929-943). Each of these technologies comes with its own benefits and limitations and may be thought of as complimentary to one another depending on the application. Kaede represents the first discovery of a photoactivatable fluorescent protein and much progress has been made in recent years with purely genetically encoded approaches (Ando et al., 2002, Proc. Natl. Acad. Sci. USA 99: 12651-12656; Tomura et al., 2008, Proc. Natl. Acad. Sci. USA 105: 10871-10876; Hayashi et al., 2007, J. Mol. Biol. 372: 918-926; Dittrich et al., 2005, Biophys. J. 89: 3446-3455). Small molecule photoactivatable probes have also found widespread use and they generally rely on a small number of photoactivation mechanisms such as photoisomerization (Cho et al., 2009, Tet. Lett. 50: 4769-4772; Gagey et al., 2007, J. Am. Chem. Soc. 129: 9986-9998), photo-uncaging (Faal et al., 2015, Mol. Biosyst. 11: 783-790; Ballister et al., 2014, Nat. Comm. 5: 5475; Kobayashi et al., 2012, J. Am. Chem. Soc. 134: 11153-11160), photodecomposition of azide (Lord et al., 2008, J. Am. Chem. Soc. 130: 9204-9205), or photoclick reactions (Lim and Lin, 2011, Accounts Chem. Res. 44: 828-839; Yu et al., 2011, J. Am. Chem. Soc. 133: 11912-11915; An et al., 2013, Org. Lett. 15: 5496-5499), to name a few. These strategies primarily depend on the activation from an initial non-fluorescent state to a fluorescent state.
Applications for fluorophores range from medicinal therapies to bioimaging to materials science and beyond (Kamkaew et al., 2013, Chem. Soc. Rev. 42: 77-88; Huang et al., 2012, Org. Lett. 14: 2594-2597; Zhang et al., 2011, Angew. Chem. Int. Ed. Eng. 50: 11654-11657; Yuan et al., 2012, J. Am. Chem. Soc. 134: 13510-13523; Lu et al., 2011, Angew. Chem. Int. Ed. Eng. 50: 11658-11662; Pansare et al., 2012, Chem. Mat. 24: 812-827; Law, 1993, Chem. Rev. 93: 449-486). Manipulation of known scaffolds such as cyanine (Mishra et al., 2000, Chem. Rev. 100: 1973-2011), BODIPY (Kamkaew et al., 2013, Chem. Soc. Rev. 42: 77-88; Lu et al., 2011, Angew. Chem. Int. Ed. Eng. 50: 11658-11662; Zhao and Carreira, 2005, Angew. Chem. Int. Ed. Eng. 44: 1677-1679, Loudet and Burgess, 2007, Chem. Rev. 107: 4891-4932), rhodamine (Zheng et al., 2013, Chem. Comm. 49: 429-447), squaraines (Avirah et al., 2012, Org. Biomol. Chem. 10: 911-920), porphyrins (Wang et al., 2013; Curr. Org. Chem. 17: 3078-3091), and napthylenediimides (Yuan et al., 2013, Chem. Soc. Rev. 42: 622-661) are commonplace in the literature.
Live cellular imaging of is one of the most powerful tools for increasing understanding of biological behavior, and its limitations primarily stem from a need for more effective imaging agents. A good subcellular stain incorporates an effective fluorophore and localizes with specificity. Designing a stain a priori involves the daunting challenge of trying to predict the various properties that are inherently associated with such efficacy, and also involves achieving and improving a multitude of properties:                1) Fluorescence        2) Water solubility and cell permeability        3) Non-toxicity        4) Red-shifted excitation and emission maxima        5) Stokes shift        6) Resistance to photobleaching        7) Compatibility with commonly used laser lines        8) Brightness        9) Site specific localization        
Moreover, in order to study the dynamics of biological processes, prolonged exposure to exciting light is sometimes unavoidable. Anti-fading agents have been developed to reduce bleaching. However, most are still fluorophore-dependent and mostly used for fixed specimens. As a result, photostability has become one of the most desirable properties of live-cell imaging dyes.
It is extremely difficult to make a compound that meets all of these requirements, and heretofore the ability to accurately predict most of these properties for a given molecule is also lacking. Even if such predictions were straightforward, many of these designs would also be synthetically undemanding to assemble and tune for greater efficacy. As such, it would be of great benefit to the bioimaging field if new imaging agents could be generated and evaluated much more rapidly.
Mitochondria are essential organelles in energy production and also involve in many other cellular activities such as differentiation, proliferation, and apoptosis. Some human neurodegenerative diseases like Alzheimer's and Parkinson's have also been correlated to mitochondrial dysfunction. Mitochondria morphologies can vary from rounded fragments to complex interconnected networks through fission and fusion. Visualizing and monitoring these dynamic organelles will shed light to many important cellular processes.
There is a need in the art for rapid and efficient methods for identifying new imaging agents. The present invention addresses and meets these needs.